The condition of membrane lipids which compose the biomembranes of organisms changes from liquid crystal to solid in accordance with the decrease in external temperatures. This change is called "phase separation". The phase separation involve change in properties of biomembranes. It is believed that membrane lipids lose the selectivity of mass permeability in solid conditions, making it impossible for biomembranes to carry out their essential functions and that, as a result, cells receive an injury (low temperature injury).
The phase transition temperatures of membrane lipids, at which the condition of the membrane lipids changes from liquid crystal to solid, are chiefly dependent on the degree of unsaturation (the number of double bonds in carbon chains) of fatty acid acyl groups bound to lipids. A lipid molecular species in which two bound fatty acid acyl groups are both saturated fatty acid residues has a higher phase transition temperature than room temperature, whereas a lipid molecular species having at least one double bond in bound fatty acid acyl groups has a phase transition temperature below about 0.degree. C. (Santaren, J. F. et al., Biochim. Biophys. Acta, 687:231, 1982).
In general, the position of a double bond in a fatty acid is indicated after the symbol ".DELTA." by the number of carbons from the carboxyl terminus to the carbon having the double bond. The total number of double bonds is indicated after a colon following the total number of carbons. For example, linoleic acid is designated as 18:2 .DELTA.9,12, which is represented by the following structural formula: EQU CH.sub.3 (CH.sub.2).sub.4 CH.dbd.CHCH.sub.2 CH.dbd.CH(CH.sub.2).sub.7 COOH
In some cases, the position of a double bond is indicated after the symbol ".omega." by the number of carbons from the methyl terminus of a fatty acid to the carbon having the double bond.
Among the membrane lipids of higher plants, only phosphatidylglycerol (PG) contains a relatively large number of saturated molecular species and it has been strongly suggested that the phase transition of PG is responsible for low temperature injury in plants (Murata, N. et al., Plant Cell Physiol., 23:1071, 1982; Roughan, P. G., Plant Physiol., 77:740, 1985) and that the molecular species composition of PG is determined by the substrate specificity of glycerol-3-phosphate acyl transferase (hereinafter referred to as "ATase") present in chloroplasts (Frentzen, M. et al., Eur. J. Biochem., 129:629, 1983; Murata, N., Plant Cell Physiol., 24:81, 1983; Frentzen, M. et al., Plant Cell Physiol., 28:1195, 1988).
Nishizawa et al. showed that if an ATase gene obtained from Arabidopsis thaliana Heynhold, a plant resistant to chilling, was introduced and expressed in tobacco, the content of saturated molecular species of PG decreased, thereby imparting a higher chilling resistance to the tobacco than when it was of a wild type (PCT/JP92/00024, 1992). However, the ATase exists originally in plants and even if a large amount of an exogenous ATase is expressed in plants, it will compete inevitably with the endogenous ATase and its effect is therefore likely to be diluted. For example, the content of saturated molecular species of PG was about 28% in the leaf of a clone which expressed the largest amount of ATase from Arabidopsis thaliana Heynhold out of the created tobacco transformants, which content was lower by about 8% than in the wild-type tobacco and higher by about 8% than in the wild-type Arabidopsis thaliana Heynhold (PCT/JP92/00024, 1992).
In general, the majority of acyl-ACP produced in plastids consists of 16:0-ACP and 18:1-ACP, and their proportions are believed to be equal. In some tissues, the proportions of 16:0-ACP and 18:0-ACP may be higher than that of 18:1-ACP (Toriyama, S. et al., Plant Cell Physiol., 29:615, 1988). In these tissues, it may be difficult to reduce satisfactorily the content of saturated molecular species by using an exogenous ATase.
The composition of membrane lipids in photosynthetic cyanobacteria (blue-green algae) is similar to that of lipids in membrane systems composing higher plant's chloroplasts (Murata, N. et al., in "The Biochemistry of Plants", Academic Press, 1987). In blue-green algae, the degree of unsaturation of fatty acids bound to membrane lipids is controlled by enzymes capable of desaturating lipid-bound fatty acids. It is known that Anacystis nidulans (Synechococcus PCC 7942) which can introduce only one double bond into lipid-bound fatty acids is sensitive to chilling (Ono, T. et al., Plant Physiol., 67:176, 1981), whereas Synechocystis PCC6803 which can introduce at least two double bonds is resistant to chilling (Wada, H. et al., Plant Cell Physiol., 30:971, 1989).
All the desaturases of fatty acids in blue-green algae react with lipids as substrates to introduce a double bond into lipid-bound fatty acids. Therefore, a cis-type double bond can be introduced into fatty acids such as PG, SQDG, MGDG and DGDG in membrane lipids of blue-green algae, which are composed of 16:0/16:0- and 18:0/16:0-saturated molecular species (Murata, N. et al., in "The Biochemistry of Plants", Academic Press, 1987). In this respect, blue-green algae are very much different from higher plants that have fatty acid desaturases capable of introducing a double bond into stearoyl-ACP (18:0-ACP) at the .DELTA.9 position and which never introduce a cis-type double bond into PG or SQDG after the synthesis of these lipids, which are composed of 16:0/16:0- (and a little amount of 18:0/16:0-) as saturated molecular species.
At present, it is known that the introduction and expression of the .DELTA.12 desaturase gene of Synechocystis PCC6803 in Anacystis nidulans enables the production of 16:2 .DELTA.9,12 which is not inherently present in the Anacystis nidulans, thereby imparting a chilling resistance to the Anacystis nidulans which is essentially chilling-sensitive (Wada, H. et al., Nature, 347:200, 1990).
Genes so far obtained for desaturases of blue-green algae include a .DELTA.6 desaturase gene (Reddy, A. S. et al., Plant Mol. Biol., 27:293, 1993) and a .DELTA.12 desaturase gene (Wada, H. et al., Nature, 347:200, 1990). However, the .DELTA.6 and .DELTA.12 desaturases cannot desaturate fatty acids at the .DELTA.6 and .DELTA.12 positions, respectively, unless a double bond is introduced at the .DELTA.9 position. Moreover, .DELTA.15 desaturase cannot desaturate fatty acids at the .DELTA.15 position unless the fatty acids are desaturated at both the .DELTA.9 and 12 positions. Hence, if the genes for enzymes capable of desaturating fatty acids at the .DELTA.9 position are introduced and expressed in higher plants, they should be able to reduce the content of saturated molecular species in higher plants and thereby impart a chilling resistance to the higher plants. However, no gene for enzymes capable of desaturating fatty acids at the .DELTA.9 position has been obtained until now.
Therefore, an object of the present invention is to provide genes for enzymes capable of desaturating fatty acids at the .DELTA.9 position and polynucleotides containing part of said genes.
Another object of the present invention is to provide vectors which contain genes for enzymes capable of desaturating fatty acids at the .DELTA.9 position or polynucleotides containing part of the genes.
A further object of the present invention is to provide plant cells and plants which are transformed with genes for enzymes capable of desaturating fatty acids at the .DELTA.9 position or polynucleotides containing part of the genes.